Tadaaki Hirose

Structural homology of Torpedo californica acetylcholine receptor subunits

The nicotinic acetylcholine receptor (AChR) from the electroplax of the ray Torpedo californica is composed of five subunits present in a molar stoichiometry of α2βγδ (refs 1–3) and contains both the binding site for the neurotransmitter and the cation gating unit (reviewed in refs 4–6). We have recently elucidated the complete primary structures of the α-, β- and δ-subunit precursors of the T. californica AChR by cloning and sequencing cDNAs for these polypeptides7,8. Here, we report the whole primary structure of the γ-subunit precursor of the AChR deduced from the nucleotide sequence of the cloned cDNA. Comparison of the amino acid sequences of the four subunits reveals marked homology among them. The close resemblance among the hydrophilicity profiles and predicted secondary structures of all the subunits suggests that these polypeptides are oriented in a pseudosymmetric fashion across the membrane. Each subunit contains four putative transmembrane segments that may be involved in the ionic channel. The transmembrane topology of the subunit molecules has also been inferred.

Primary structure of porcine cardiac muscarinic acetylcholine receptor deduced from the cDNA sequence

The complete amino acid sequence of the porcine cardiac muscarinic acetylcholine receptor has been deduced by cloning and sequencing the cDNA. The tissue location of the RNA hybridizing with the cDNA suggests that this muscarinic receptor species represents the M2 subtype.

Primary structure of the α-subunit of bovine adenylate cyclase-stimulating G-protein deduced from the cDNA sequence

The primary structure of the α‐subunit of the adenylate cyclase‐stimulating G‐protein (Gs) has been deduced from the nucleotide sequence of cloned DNA complementary to the bovine cerebral mRNA encoding the polypeptide. Comparison of the amino acid sequences of the α‐subunits of Gs and transducin reveals that some of the highly conserved regions show sequence homology with elongation factor‐Tu and ras p21 proteins and correspond to functional regions of guanine nucleotide‐binding proteins.

Primary structure of the β‐subunit of Torpedo californica (Na+ + K+)‐ATPase deduced from the cDNA sequence

DNA complementary to the Torpedo californica electroplax mRNA coding for the β‐subunit of (Na+ + K+)‐ATPase has been cloned by screening a cDNA library with an oligodeoxyribonucleotide probe. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 305 amino acid residues (including the initiating methionine). The transmembrane topology and the potential N‐glycosylation sites of this polypeptide are discussed.

Molecular Genetic Analysis of Myelin-Deficient Mice: Shiverer Mutant Mice Show Deletion in Gene(s) Coding for Myelin Basic Protein

Abstract: The gene expression of myelin basic proteins (MBPs) in shiverer mutant mice was investigated by the Northern and Southern hybridization techniques. In the control mice RNA molecules from the brains which were about 2,300 nucleotides in length were hybridized to cDNA of 1.8 kb encoding for a mouse MBP, but RNA from the brains of 3‐week‐old shiverer mutant mice contained no detectable amount of MBP transcripts hybridizing to this probe. Moreover the shiverer mutant mice lost several restriction fragments that hybridized to the same probe in the control mice when each of the five restriction enzymes, i.e., HindIII, PstI, PvuII, AccI, and StuI, was used. These data suggest that the shiverer mutation may correspond to the deletion of a large portion of MBP exon(s) in the gene, and this deletion causes inefficient transcription leading to the depletion of MBPs in the myelin and the dysmyelination observed in these mice.

Analysis of the complete nucleotide sequence of the group IV RNA coliphage SP.

We report the nucleotide sequence of the Group IV RNA bacteriophage SP. The entire sequence is 4276 nucleotides long. Four cistrons have been identified by comparison with the related Group III phage Q beta. The maturation protein contains 449 amino acids, the coat protein contains 131 amino acids, the read-through protein contains 330 amino acids and the replicase beta-subunit contains 575 amino acids. SP is 59 nucleotides longer than Q beta. We have analyzed both sequence and structural conservation between SP and Q beta and shown that the sequences for the coat and central region of the replicase are strongly conserved between the two genomes. We also show that the S and M replicase binding sites of Q beta are strongly conserved in SP. Interestingly, the base composition of SP and Q beta differ significantly from one another, and most of the differences can be accounted for by a strong preponderance of U in the third position of each codon of Q beta relative to SP. We also compare conserved hairpins associated with potential coat protein and replicase binding sites.

Substitutions of Artificial Amino Acids O-Methyl-Thr, O-Methyl-Asp, S-Methyl-Cys, and 3-Amino-Ala for Thr-252 of Cytochrome P-450cam: Probing the Importance of the Hydroxyl Group of Thr-252 for Oxygen Activation

In the monooxygenation reaction catalyzed by cytochrome P-450cam (P450cam), Thr-252, a hydroxyl amino acid at the active site, is essential for the reductive cleavage of the O-O bond of oxygen: the hydroxyl (OH) group of Thr has been proposed to serve as an acid catalyst for the O-O bond scission. In this study, four different artificial amino acids were incorporated into the 252-position to verify the role of the OH group. The catalytic activities of the mutant enzymes suggest that the OH group does not function as the catalyst. Then, we propose that the OH group serves as an anchor of an acid catalyst and facilitates its catalytic action to cleave the O-O bond; water is possibly the catalyst.